The transfer of DNA and or proteins to susceptible hosts is critical to bacterial pathogenesis. Type IV secretion systems (T4SS) are used by gram-negative bacteria to transport DNA and or proteins into eukaryotic cells and cause disease. T4SS components are highly conserved among diverse bacteria. The Agrobacterium T4SS is one of the most extensively studied and serves as the prototype. Thus, research to characterize T4SS in Agrobacterium has wide application to designing strategies to block such transfer in medically important diseases. Though the transfer of DNA and or proteins is known to be critical to pathogenesis, the exact mechanism of this transfer remains largely unknown. The long term objectives of the proposed research are to characterize the assembly and function of T4SS using a variety of molecular, genetic, biochemical and cell biological methods. Previously published reports suggest that the T4SS localizes to the poles of the bacterial cell;this localization suggested there would be one point of contact with the host cell to transfer DNA and proteins. However, I have new exciting results to suggest that the T4SS localizes both to the poles and in a helical pattern around the circumference of the bacterial cell. This helical localization of multiple T4SS gives the bacteria a greater likelihood of making productive contact with the host during DNA and protein transport. Specific aim 1 will analyze this localization pattern in detail by following the kinetics of helical array formation, by determining its genetic requirements, and what happens to this localization pattern following contact with plant host cells. The helical localization of the T4SS resembles the helical stnjcture of cytoskeletal components in bacteria. This observation leads us to propose that the cytoskeleton of Agrobacteria has a role In the localization of the T4SS. In specific aim 2 will investigate the potential role of the cytoskeleton on T4SS assembly and function.